Modified Hemoglobin Solutions Research Articles

Normalization of hemoglobin-based oxygen carrier-201 induced vasoconstriction: targeting nitric oxide and endothelin.

Hemoglobin-based oxygen carrier (HBOC)-201 is a cell-free modified hemoglobin solution potentially facilitating oxygen uptake and delivery in cardiovascular disorders and hemorrhagic shock. Clinical use has been hampered by vasoconstriction in the systemic and pulmonary beds. Therefore, we aimed to 1) determine the possibility of counteracting HBOC-201-induced pressor effects with either adenosine (ADO) or nitroglycerin (NTG); 2) assess the potential roles of nitric oxide (NO) scavenging, reactive oxygen species (ROS), and endothelin (ET) in mediating the observed vasoconstriction; and 3) compare these effects in resting and exercising swine. Chronically instrumented swine were studied at rest and during exercise after administration of HBOC-201 alone or in combination with ADO. The role of NO was assessed by supplementation with NTG or administration of the eNOS inhibitor Nω-nitro-l-arginine. Alternative vasoactive pathways were investigated via intravenous administration of the ETA/ETB receptor blocker tezosentan or a mixture of ROS scavengers. The systemic and to a lesser extent the pulmonary pressor effects of HBOC-201 could be counteracted by ADO; however, dosage titration was very important to avoid systemic hypotension. Similarly, supplementation of NO with NTG negated the pressor effects but also required titration of the dose. The pressor response to HBOC-201 was reduced after eNOS inhibition and abolished by simultaneous ETA/ETB receptor blockade, while ROS scavenging had no effect. In conclusion, the pressor response to HBOC-201 is mediated by vasoconstriction due to NO scavenging and production of ET. Further research should explore the effect of longer-acting ET receptor blockers to counteract the side effect of hemoglobin-based oxygen carriers.NEW & NOTEWORTHY Hemoglobin-based oxygen carrier (HBOC)-201 can disrupt hemodynamic homeostasis, mimicking some aspects of endothelial dysfunction, resulting in elevated systemic and pulmonary blood pressures. HBOC-201-induced vasoconstriction is mediated by scavenging nitric oxide (NO) and by upregulating endothelin (ET) production. Pressor effects can be prevented by adjuvant treatment with NO donors or direct vasodilators, such as nitroglycerin or adenosine, but dosages must be carefully monitored to avoid hypotension. However, hemodynamic normalization is more easily achieved via administration of an ET receptor blocker.

Importância dos carreadores de oxigênio livre de células

Os procedimentos necessários para redução de efeitos adversos associados à transfusão de sangue, em especial aqueles decorrentes da transmissão de agentes infecciosos e da aloimunização leucócito-mediada têm impacto nos custos de produção de hemocomponentes. Paralelamente, as necessidades transfusionais têm aumentado globalmente, ficando evidente a necessidade de um substituto seguro e amplamente disponível para o sangue, chamado de sangue artificial ou de substituto do sangue. Visto que o seu desenvolvimento tem se concentrado na função de carrear oxigênio aos tecidos, daí utilizarmos, nesse texto, a denominação "Carreadores de oxigênio livre de células". Atualmente, dois tipos de carreadores de oxigênio livre de células têm sido testados: as soluções de hemoglobina modificadas (de origem humana ou bovina) e os perfluorocarbonos (PFCs). Entretanto, esses produtos não são isentos de efeitos adversos e um grande número de pesquisas clínicas está em andamento para testar sua eficácia e segurança. O maior conhecimento desses carreadores de oxigênio livre de células e seus mecanismos de ação permitiu que aplicações outras, até mesmo não clínicas, estivessem em teste com as novas gerações desses produtos, expandindo assim as fronteiras da medicina transfusional.

Chemically modified porcine hemoglobins and their biological properties.

Hemoglobin cross-linked with small molecular modifiers turns out to be more stable. Modifications of proteins with polyethylene glycol (PEG) have been proven to enlarge the molecular size of proteins, to prolong their retention time in the circulation as well as blunt immune reactions. In the present study, the optimal conditions for porcine hemoglobin (pHb) modification with bis (3, 5-dibromosalicyl) fumarate (DBBF) and PEG were evaluated. The derivative of DBBF cross-linked pHb (DBBF-pHb) showed improved oxygen affinity and the ability to resist the dissociation of the alpha2beta2 tetramer compared with the natural protein. DBBF-pHb was then bound to the activated PEG. The results indicated that the pHb modified with DBBF and PEG had more stable tetrameric conformation with a molecular weight of 107000. Their oxygen half-saturation pressure (P50) is around 3.33 kPa, which approximates the physiological P50 of human red blood cells. Both routine and reinforced immunizing methods were adopted to study the immunogenicity of modified products and the results showed that the products had very low immunogenicity evaluated by enzyme-linked immunoadsordent assay (ELISA). Somewhat beneficial effects were shown in the treatment of hemorrhagic shock where modified hemoglobin solutions were used as resuscitation fluids in the hemorrhagic shock Sprague-Dawley (SD) rats model.

Doping with Artificial Oxygen Carriers

There is a long history of science seeking to develop artificial substitutes for body parts damaged by disease or trauma. While defective teeth and limbs are commonly replaced by imitations without major loss of functionality, the development of a substitute for red blood cells has proved elusive. There is a permanent shortage of donor blood in western societies. Nevertheless, despite whole blood transfusions carrying measurable risks due to immunogenicity and the transmission of blood-borne infectious diseases, red blood cells are still relatively inexpensive, well tolerated and widely available. Researchers seeking to develop products that are able to meet and perhaps exceed these criteria have responded to this difficult challenge by adopting many different approaches. Work has focussed on two classes of substances: modified haemoglobin solutions and perfluorocarbon emulsions. Other approaches include the creation of artificial red cells, where haemoglobin and supporting enzyme systems are encapsulated into liposomes. Haemoglobin is ideally suited to oxygen transport when encased by the red cell membrane; however, once removed, it rapidly dissociates into dimers and is cleared by the kidney. Therefore, it must be stabilised before it can be safely re-infused into humans. Modifications concomitantly alter the vascular half-life, oxygen affinity and hypertensive characteristics of raw haemoglobin, which can be sourced from outdated blood stores, genetically-engineered Escherichia coli or even bovine herds. In contrast, perfluorocarbons are entirely synthetic molecules that are capable of dissolving oxygen but biologically inert. Since they dissolve rather than bind oxygen, their capacity to serve as a blood substitute is determined principally by the oxygen pressure gradients in the lung and at the target tissue. Blood substitutes have important potential areas of clinical application including red cell replacement during surgery, emergency resuscitation of traumatic blood loss, oxygen therapeutic applications in radiography (oxygenation of tumour cells is beneficial to the effect of certain chemotherapeutic agents), other medical applications such as organ preservation, and finally to meet the requirements of patients who cannot receive donor blood because of religious beliefs. Given the elite athlete's historical propensity to experiment with novel doping strategies, it is likely that the burgeoning field of artificial oxygen carriers has already attracted their attention. Scientific data concerning the performance benefits associated with blood substitutes are virtually nonexistent; however, international sporting federations have been commendably proactive in adding this category to their banned substance lists. The current situation is vulnerable to exploitation by immoral athletes since there is still no accepted methodology to test for the presence of artificial oxygen carriers.

Volume expansion with modified hemoglobin solution, colloids, or crystalloid after hemorrhagic shock in rabbits: effects in skeletal muscle oxygen pressure and use versus arterial blood velocity and resistance.

Therapeutic goals for hemorrhagic shock resuscitation are the increase of cardiac output and oxygen delivery. The possibility exists that because of microcirculatory effects, different volume expanders result in different tissue oxygen delivery and oxygen use. In a rabbit model of resuscitation from hemorrhagic shock (50% blood loss), we compared the effects of an hemoglobin-based O2-carrying solution (HbOC) with those elicited by albumin, hydroxyethyl starch (HES), or saline on systemic hemodynamics, skeletal muscle O2 pressure (PtiO2), and interstitial concentration of lactate (LACi) through the combined implantation of a microdialysis probe and a sensitive O2 electrode into the hind limb. Hemorrhagic shock induced a 50% decrease in mean arterial pressure (MAP), femoral artery blood flow (BF), and PtiO2. After resuscitation, there were statistically significant differences among the volume expanders. The increase in MAP was faster with HbOC and colloids, and slower with saline, mainly obtained by vasoconstriction for HbOC and by increased BF with albumin and HES. The maximum MAP values were significantly higher for HbOC compared with the other volume expanders. HbOC and colloids induced a faster increase in PtiO2 as compared with saline, but maximum PtiO2 values were not different among the volume expanders. Tissue oxygen use as estimated by LACi increased transiently at the beginning of volume expansion with similar maximum values. Animals resuscitated with saline had significantly higher LACi concentrations after the onset of volume expansion as compared with HbOC but not with colloids. Our results demonstrate that there are measurable differences in MAP and BF upon resuscitation with the four different solutions and there is a slower increase in tissue PtiO2 with saline than with colloids associated with significantly increased LACi consistent with delayed reoxygenation upon resuscitation with saline.

Blood Substitutes

Three classes of materials have been studied as potential blood substitutes: modified hemoglobin solutions, perfluorocarbon emulsions, and liposome-encapsulated hemoglobin. The first two have reached phase III clinical trials, while the third remains in the preclinical state of testing. Hemoglobin is a highly active molecule; hence, modification has been required to avoid potential deleterious effects. Although there has been considerable progress toward bringing such a product to the clinical setting, its development has challenged our understanding of oxygen delivery and use. The perfluorocarbon emulsions have been studied primarily for roles other than as equivalents of conventional banked units of RBCs for transfusion. The study of these molecules has added to our understanding of basic physiologic processes.

Augmented acute normovolemic hemodilution (A-ANH TM ) -

Augmented Acute Normovolemic Hemodilution serves to reduce the need for allogeneic blood transfusions in patients undergoing high blood loss surgery. Augmented acute normovolemic hemodilution has 3 phases: First, acute normovolemic hemodilution prior to surgical blood loss, second, administration of an artificial O2 carrier such as a modified hemoglobin solution or perflubron emulsion during periods of low endogenous hemoglobin concentration during surgery and third, re-transfusion of autologous blood after the operation. Efficacy remains to be ultimately proven in clinical trials but mathematical modeling indicates that clinically significant reductions in allogeneic blood transfusions are likely.

Hemoglobin-based oxygen carriers, blood substitutes, and their relevance to high-volume blood loss

T ECHNICALLY, a substitute is a substance that can effectively replace most functions of human blood. However, modified hemoglobin solutions and perfluorocarbons have also been referred to as blood substitutes. Because these recently developed solutions can perform only selected functions of blood, they are more accurately referred to as oxygen-carrying volume expanders. Hemoglobin-based oxygen carriers (HBOCs) are modified hemoglobin solutions or hemoglobin packaged into liposomes, which are able to deliver oxygen to tissues. A hemoglobin therapeutic is a hemoglobin solution optimized through chemical modification to bring about certain pharmacological and therapeutic effects. Hemoglobin therapeutics may possess a combination of therapeutically active properties such as capacity, favorable theologic properties, and pressor action.

Les transporteurs d'oxygène à base d'hémoglobine

The modified hemoglobins are not true blood substitutes because they do not possess the complex functions of erythrocytes. They are oxygen carriers only with a short intravascular life, adapted for temporary use. Three types of modified hemoglobin solutions have been designed, prepared from human or bovine hemoglobin or by genetic engineering. These hemoglobins are highly purified to eliminate trace amounts of stroma, lipids and endotoxins, which are responsible for acute toxicity. They are modified by internal cross linking between the monomers, or by binding to macromolecules. Afterwards, they can be polymerized or encapsulated in liposomes. The purpose of these modifications is to increase oxygen delivery to tissue, to reduce the dissociation into monomers and to guard against oxydation into methemoglobin. The modified hemoglobin solutions are essentially used in hemorrhagic shock and perioperative hemodilution. These oxygen carriers could be used by some athlets to increase their results in particular during endurance sports. But these hemoglobins are different enough from physiologic hemoglobins (normal or pathological), so they could be easily detected in blood samples.

Physiological properties of blood substitutes.

Blood substitutes (modified hemoglobin solutions, perfluorocarbon emulsions) serve as artificial oxygen carriers and are alternatives to blood transfusions. Hemoglobin solutions mimic the sigmoidal oxygen dissociation curve of natural blood. Perfluorocarbon emulsions exhibit a linear relation between PO2 and dissolved oxygen. The most advanced substances may enter medicine in few years.

Current status of artificial oxygen carriers

Artificial oxygen carriers may be grouped into modified hemoglobin solutions and fluorocarbon emulsions. In animal experiments, both have been shown to be efficacious in improving tissue oxygenation and as substitutes for blood transfusions. Advantages and disadvantages are being discussed in this article as well as the latest steps in the clinical development.

Administration of polymerized bovine hemoglobin improves survival in a rat model of traumatic shock.

The ability of purified and modified hemoglobin solutions to expand blood volume and act as oxygen and carbon dioxide transporters with no apparent toxic side effects has lead to an increased interest in their utilization in shock conditions. Although hemoglobin solutions have been shown to provide beneficial effects in endotoxic and hemorrhagic shock, they have not been studied in whole body traumatic injury. We investigated the effects of a polymerized bovine hemoglobin solution (HBOC-201) in a rat model of traumatic shock. Pentobarbital-anesthetized rats subjected to Noble-Collip drum trauma developed a shock state characterized by marked hypotension, a survival time of 102 +/- 20 min, significant increases in intestinal myeloperoxidase (MPO) activity and splanchnic vascular endothelial dysfunction characterized by an impaired vasorelaxation of the superior mesenteric artery (SMA) to endothelium-dependent vasodilators. Traumatized rats were treated with HBOC-201 or an equal volume of vehicle corresponding to 5, 10 or 15% of the calculated blood volume. Treatment with HBOC-201 10 min posttrauma prolonged survival, normalized mean arterial blood pressure and attenuated endothelial dysfunction of the SMA. However, administration of HBOC-201 failed to significantly attenuate increases in intestinal MPO activity following trauma. No beneficial effects were observed with the administration the vehicle. These data indicate that HBOC-201 exerts significant beneficial effects in traumatic shock states by normalizing systemic blood pressure and attenuating vascular endothelial dysfunction. HBOC-201 may serve as a useful adjunctive agent in the early treatment of trauma.

HEMODYNAMIC EFFECTS OF MODIFIED HEMOGLOBIN IN OVINE SEPSIS ARE ANTAGONIZED BY L-ARGININE

S125 Introduction: Modified hemoglobin solutions like pyridoxalated hemoglobin polyoxyethylene (PHP) reverse the hyperdynamic circulation in sepsis. [1] They bind nitric oxide (NO), a mediator for the hemodynamic changes in sepsis. [2,3] Their use is associated with pulmonary hypertension. We studied the effect of L-arginine, a NO precursor, after PHP administration in sepsis. Methods: Sheep were instrumented with arterial, venous, left atrial, and pulmonary catheters. After 5 days Ps. aeruginosa (6x106 CFU/kg/h) was infused for 32 h. After 24 hr of Ps., 20 mg/kg/h PHP was infused for the rest of the study in group PHP (n=14) and group Arg (n=6); group S (n=12) received the carrier solution. At 28h, group Arg received a bolus of 100mg/kg L-arginine followed by 500mg/kg/h for 1h. Statistical analysis by Anova. Results: Data in the graphs are presented as mean +/- SEM, closed squares: group Arg, triangles: group PHP; circles: group S. (Figure 1, Figure 2, and Figure 3)Figure 1Figure 2Figure 3Discussion: PHP increased systemic and pulmonary vascular resistance. L-arginine rapidly decreased the resistances to their 24hr levels after Ps. The administration of large amounts of L-arginine could cause a rise in NO production that exceeded the binding capacity of the low dose PHP infusion. L-arginine may be useful as a rapidly acting agent to reverse undesirable vasoconstriction after administration of hemoglobin in sepsis. This study was in part supported by Apex Bioscience. Inc.

An Improved Process for the Production of Sterile Modified Haemoglobin Solutions

The process for manufacturing bulk quantities of sterile solutions of human haemoglobin (Hb) cross-linked between the alpha chains (ααHb) with bis(3,5-dibomosalicyl) fumarate (DBBF) was modified to: (1) improve product purity; (2) increase product yield; (3) eliminate non-United States Pharmacopoeia materials; (4) reduce reagent costs; and (5) reduce production time. These process modifications were the result of increased scientific understanding of the Hb cross-linking chemistry and were in the form of engineering and procedure controls that reflect current good manufacturing practices (cGMP). Purity, as reflected in the fractional yield of the desired ααHb product, has increased from 60% to 90+% of total Hb, and uncross-linked Hb was virtually eliminated. Impurities such as pyrogens, methaemoglobin, phospholipid, and free iron were reduced. The net yield of ααHb was increased from 33% to 58% of starting Hb content. Production time, the use of overtime, the consumption of expensive reagents and filters, and losses because of contamination have all been reduced. As a result, cost per gram of ααHb produced has decreased 60%. With this improved process, efficient production of very pure ααHb is possible.

Expression of adhesion molecules and von Willebrand factor in human coronary artery endothelial cells incubated with differently modified hemoglobin solutions.

Previous studies have established a linkage between free Hb molecules and the production of inflammatory mediators by the reticuloendothelial cells. An important aspect of the endothelial response to the inflammatory stimuli is the expression of adhesion molecules on the luminal surface. Therefore, the present study was designed to investigate the effects of various free-Hb based oxygen carrying solutions on the intracellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1) and also von Willebrand factor (vWF) expression by human endothelium. Human coronary artery endothelial cells (HCAEC) were cultured on glass coverslips until they reached confluence, then incubated for 18 hours with endothelial basal medium (EBM) supplemented with 5% FBS and a 0.1 mmol or 0.2 mmol of the bovine Hb solutions: 1) pure unmodified bovine Hb (UHb); 2) modified bovine Hb solution (Hb-PP-GSH) prepared according to our newly developed procedure (U.S. Patent No. 5,439,882); and 3) modified bovine Hb solution polymerized with glutaraldehyde (GLUT-Hb). The HCAECs were also incubated with EBM (negative control) and EBM containing bacterial endotoxins in a concentration of 50 EU/ml (positive control). After treatment, cells were exposed to primary antibodies; anti-human ICAM-1, anti-human VCAM-1 or anti-human vWF, and consequently to the secondary antibody (fluorescein isothiocyanate-conjugated F(ab)2). Immunofluorescence analysis revealed different expressions of ICAM-1 and VCAM-1 on the surface membranes of variously treated cells. Although negative control cells had an undetectable level of adhesion molecules, the positive control cells, activated by endotoxin, exhibited high immunoreactivity for ICAM-1 and VCAM-1. The Hb's treated cells demonstrated differing degrees of activation. An insignificant expression of ICAM-1 was observed in HCAEC, following treatment with a 0.1 or 0.2 mmol of Hb-PP-GSH and 0.1 mmol of UHb. Cell treated with 0.2 mmol of UHb and both concentrations of GLUT-Hb demonstrated a massive expression of this adhesion molecule. A similar effects was observed during induction of VCAM-1. While a lack of expression was noted with both concentrations of Hb-PP-GSH and 0.1 mmol of UHb, the GLUT-Hb stimulated significant VCAM-1 induction at all tested concentrations. Immunofluorescence analysis confirmed the expression of vWF uniformly in HCAEC from the different experimental groups. The data suggest, vWF expression was unaffected by all but the GLUT-Hb treatment. In conclusion, the Hb stimulatory activity toward ICAM-1 and VCAM-1 inductions were related with the type of Hb chemical modification method. Although modification of Hb with glutaraldehyde potentiates adhesion molecules expression, our novel Hb modification procedure, which comprises intramolecular cross-linking with o-adenosine triphosphate and intermolecular with o-adenosine, and combined with reduced glutathione, apparently prevents these inflammatory events.

Safety evaluation of a polymerized hemoglobin solution in a murine infection model

Several investigators have observed that free hemoglobin may increase the mortality rate in experimental Escherichia coli peritonitis in animals. This effect is probably mediated by the heme moiety of hemoglobin, but the mechanism remains controversial. Free hemoglobin might impair neutrophil function, and it might serve as a source of iron, which is necessary for bacterial replication. Several modified hemoglobin solutions, developed as blood substitutes, are currently being tested in clinical studies, but concern exists that these solutions may have the potential to exacerbate a bacterial infection. At the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, a blood substitute based on modified hemoglobin (PolyHbXI) has been developed that has improved oxygen affinity and prolonged vascular retention. In the present study the potential risk of this solution on the promotion of infections has been evaluated. PolyHbXl was intravenously injected into mice in a clinically relevant dose of 1.5 gm/kg body weight 1 hour before intravenous administration of a sublethal number of Listeria monocytogenes, Salmonella typhimurium, E. coli, or Candida albicans organisms. PolyHbXl did not promote the proliferation of any of these microorganisms in the liver and spleen, nor did it lead to an increased mortality rate in the mice. Also, the in vitro proliferation of L. monocytogenes, S. typhimurium, and E. coli was not increased by PoIyHbXI. In conclusion, PoIyHbXI does not affect the course of infection with various microorganisms in mice, and no indication was found that this new blood substitute compromises the host defense against infections.

AN IMPROVED BLOOD SUBSTITUTE. IN VIVO EVALUATION OF ITS HEMODYNAMIC EFFECTS

The purpose of the present study was to assess the ability of an improved free hemoglobin based blood substitute to serve as a resuscitative fluid in the treatment of hemorrhagic shock. Comparison studies were performed by using blood autotransfusion as a positive control. The hemodynamic parameters studied included cardiac index, mean arterial pressure, pulse pressure, heart rate, stroke volume index, and total peripheral resistance. Tissue oxygenation was measured in the biceps femori muscle by polarography. Hemorrhagic shock (at 40% of the total blood volume) in anesthetized rats caused severe disturbances in hemodynamic parameters and tissue oxygenation. Shock was characterized by a 66% drop in cardiac index, a 67% drop in mean arterial pressure with a significant increase in total peripheral resistance, and a 78% reduction in tissue oxygenation, all lasting 30 min. Resuscitation from shock with the blood substitute was effective in restoring hemodynamic parameters, producing vasodilation, and improving tissue oxygenation. Autotransfusion with blood also restored hemodynamics. However, lower tissue oxygenation and lack of vasodilation were noted. Therefore, the modified hemoglobin solution yielded better results than blood in the resuscitation of rats after hemorrhagic shock. The vasodilatory activity and the reduction of vasoconstriction that followed hemorrhage can be primarily linked with adenosine, which possesses vasodilatory and anti-inflammatory properties, and is used in our technology as an intermolecular cross linking reagent and hemoglobin surface modifier.

Systemic Responses to Sfhs-Infusion in Hemorrhaged Dogs

Anesthetized mongrel (weight range: 16-27 Kg) dogs were prepared for monitoring hemodynamics, blood flow distribution, plasma colloid osmotic pressure and renal functional parameters at various intervals. Removal of 35 ml/Kg blood resulted in marked drop and only partial spontaneous recovery in systemic and pulmonary arterial pressures, cardiac output and organ blood flows (> 50% flow-decrements occurred in kidney, spleen, heart, gut and pancreas); plasma colloid osmotic pressure as well as urine output and creatinine clearance also fell. Group I (n = 6) of dogs was transfused after 45 minutes of hypovolemia with their own anticoagulated blood, while Group II (n = 6) received an equal volume of unmodified 6% stromafree hemoglobin solution (SFHS). Comparison of the two groups' responses to resuscitation yielded some differences. There was a significant overshoot (30 mmHg) in systemic arterial blood pressure accompanied by bradycardia in Group II only. Cardiac output recovered in both groups but was less well sustained in Group II. Cerebral blood flow rose higher and hepatic arterial flow-increment was less in Group II than in Group I; the responses to resuscitation in the other organs were comparable. Colloid osmotic pressure decreased in Group I whereas it rose immediately after resuscitation in Group II, declining thereafter with a converging trend and 30 minutes thereafter the differences were not significant between the groups. Urine excretion and creatinine clearance recovered to comparable extents in both groups, but N-acetyl-beta-D-glucosaminidase (N.A.G.) excretion rose over 10-fold higher in Group II than in Group I. These experiments have defined the response of bled animals to resuscitation with unmodified, unpurified SFHS, when compared to resuscitation with whole blood, showing a less well sustained but adequate hemodynamic and renal functional recovery while revealing indications of early renal tubular cellular injury, providing baseline comparison for testing highly purified and modified hemoglobin solutions.

Haemoglobin-based red cell substitutes: current status.

Chemically modified haemoglobin solutions represent a potential alternative to the transfusion of donor blood. The theoretical advantages of these products include an oxygen delivery potential greater than that of conventional plasma expanders, prolonged shelf-life, universal compatibility and the absence of pathogenic viruses. Principal concerns have been safety issues including renal toxicity, coagulopathy and vasoactivity. The proposed indications for these solutions are primarily resuscitation of patients in haemorrhagic shock and perioperative haemodilution during elective surgery. Three products have now undergone phase I safety trials in human subjects and phase II safety and efficacy trials are planned in the near future.

Diaspirin cross-linked hemoglobin improves neurological outcome following reversible but not irreversible CNS ischemia in rabbits.

Hemodilution using modified hemoglobin solutions may reduce ischemic central nervous system injury. Purified diaspirin cross-linked hemoglobin (DCLHb) is a cell-free hemoglobin that is intramolecularly cross-linked between the two alpha subunits, resulting in enhanced oxygen offloading to tissues and increased half-life. In the present experiments, we evaluated the ability of DCLHb to reduce neurological damage in two rabbit stroke models. In a reversible spinal cord ischemia model, ischemia of the caudal lumbar spinal cord was produced by temporary occlusion of the abdominal aorta. In an irreversible model of cerebral ischemia, plastic microspheres (50 microns) were injected into the internal carotid artery and lodged in the cerebral microvasculature. DCLHb was administered 5 minutes after initiation of ischemia as either a 10-mL/kg infusion, 10-mL/kg exchange transfusion, or a 20-mL/kg infusion. Control animals received human serum albumin that was oncotically matched to the DCLHb. In the spinal cord model, DCLHb significantly increased the duration of ischemia required to produce permanent paralysis from 27.33 +/- 8.71 minutes (mean +/- SD) in controls to 42.59 +/- 10.10 minutes in the 10-mL/kg exchange transfusion group and to 40.82 +/- 18.16 minutes in the 20-mL/kg infusion condition (P < .05). DCLHb did not significantly reduce neurological damage in the microsphere embolization model. These data suggest that cross-linked hemoglobin reduces neurological damage after reversible central nervous system ischemia and that this is not attributable to hemodilution or hypervolemia only.